Beauvericin detoxification compositions and methods

ABSTRACT

The present invention provides a bacterial microorganism having the ability to degrade or detoxify beauvericin or derivatives or analogs of beauvericin. The present invention further provides a method for detoxification of plants pre- or post-harvest using microbes having the ability to degrade or detoxify beauvericin or derivatives or analogs of beauvericin.

This application is a continuation of application Ser. No. 08/753,287filed Nov. 22, 1996, now U.S. Pat. No. 5,798,255.

TECHNICAL FIELD

The present invention relates generally to the detection and isolationof beauvericin-degrading organisms and to compositions and methods fordetoxification or degradation of beauvericin in grain. This method hasbroad application in agricultural biotechnology and crop agriculture andin the improvement of food grain quality and feed safety.

BACKGROUND OF THE INVENTION

Fungal diseases are common problems in crop agriculture. Many strideshave been made against plant diseases as exemplified by the use ofhybrid plants, pesticides and unproved agricultural practices. However,as any grower or home gardener can attest, the problems of fungal plantdisease continue to cause difficulties in plant cultivation. Thus, thereis a continuing need for new methods and materials for solving theproblems caused by fungal diseases of plants. These problems can be metthrough a variety of approaches. For example, the infectious organismscan be controlled through the use of agents that are selectivelybiocidal for the pathogens. Another method is interference with themechanism by which the pathogen invades the host crop plant. Yet anothermethod, in the case of pathogens that cause crop losses, is interferencewith the mechanism by which the pathogen causes injury to the host cropplant. Still another method, in the case of pathogens that producetoxins that are undesirable to mammals or other animals that feed on thecrop plants, is interference with toxin production, storage, oractivity.

Within the Fusarium sp. are several important pathogens of corn andother cereals in various countries. In corn, Fusarium is known to causeroot, stem and ear rot that results in severe crop reduction. Theetiology of Fusarium ear mold is poorly understood, although physicaldamage to the ear and certain environmental conditions can contribute toits occurrence (Nelson P E (1992) "Taxonomy and Biology of Fusariummoniliforme." Mycopathologia 117: 29-36). Fusarium may be isolated frommost field grown maize, when no visible mold is present. Therelationship between seedling infection and the stalk and ear diseasescaused by Fusarium is not clear. Genetic resistance to visible kernelmold has been identified. (Gendloff E, Rossman E, Casale W, Isleib T,Hart P, 1986, "Components of resistance to Fusarium ear rot in fieldcorn." Phytopathology 76: 684-688; Holley R N, Hamilton P B, Goodman MM, 1989, "Evaluation of tropical maize germplasm for resistance tokernel colonization by Fusarium moniliforme." Plant Dis 73: 578-580).The mycotoxins produced by the Fusarium species that infect plants mayaccumulate in infected plants or in stored grains, presenting serioushealth consequences for livestock, humans, and other consumers of meator other food products of such livestock. Fusarium infection has beenassociated with chronic or acute mycotoxicoses in both farm animals andman (Botallico, et al.). An important mycotoxin that has been found tobe produced by certain Fusarium sp. and has been identified in Fusariuminfected crops is beauvericin.

Beauvericin is a fungal toxin produced by various Fusarium species, aswell as the fungus Beauveria bassiana. Beauvericin is a cyclic peptide,with toxic effects on insects as well as both human and murine celllines. The activity of beauvericin is due to the ionophoric propertiesof the compound. Beauvericin is capable of forming complexes with alkalimetal cations and affects ion transport across cell membranes. Inaddition, beauvericin has been reported to be one of the most powerfulinhibitors of cholesterol acetyltransferase. Beauvericin has also beenshown to induce a type of cell death very similar to apoptosis.Circumstantial evidence further indicates that beauvericin acts inconcert with other Fusarium toxins to cause additional toxic effects(1).

Beauvericin has been reported to be found at significant levels in cornfrom Italy, Peru, and Poland (1,2,3). It is likely that beauvericin willalso be found in other areas as more studies are completed. Fusariumspecies are found in virtually all moldy as well as healthy corn. Foodsafety is an important issue for grain growers. The EuropeanCommonwealth is considering imposing limits on various mycotoxin levelsof imported grain.

There is a need in the art for novel methods with which beauvericin maybe eliminated from a plant or harvested grain. It is consideredimportant by those skilled in the art to continue to develop inventionsin order to protect the final consumer of a plant or harvested grain.The present invention provides the reagents and methodologies necessaryto ameliorate plants and harvested grains from beauvericin.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an organism having theability to degrade or detoxify beauvericin or a structurally relatedmycotoxin. The present invention may further include a mutant of thewild-type organism that has the ability to degrade or detoxifybeauvericin or a structurally related mycotoxin. The present inventionfurther provides a method for detoxification of plants pre- orpost-harvest using a microbe having the ability to degrade or detoxifybeauvericin or a structurally related mycotoxin.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery of an organism havingthe ability to degrade the mycotoxin beauvericin. The present inventionhas resulted from a search for a biological means of detoxifyingbeauvericin and comprises several bacterial species, isolated from moldywheat and residential compost, capable of growing on beauvericin as asole carbon source, degrading it partially or completely in the process.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, chemistry,and biochemistry, which are within the skill of the art. Such techniquesare explained fully in the literature. See, e.g. J. H. Langenheim and K.V. Thimann, Botany: Plant Biology and Its Relation to Human Affairs(1982) John Wiley; Cell Culture and Somatic Cell Genetics of Plants,Vol. 1 (I. K. Vasil, ed. 1984); R. V. Stanier, J. L. Ingraham, M. L.Wheelis, and P. R. Painter, The Microbial World, (1986) 5th Ed.,Prentice-Hall; O. D. Dhringra and J. B. Sinclair, Basic Plant PathologyMethods, (1985) CRC Press; Maniatis, Fritsch & Sambrook, MolecularCloning: A Laboratory Manual (1982); DNA Cloning, Vols. I and II (D. N.Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); theseries in Methods in Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Current Protocols in Molecular Biology (JohnWiley & Sons, Inc. 1996).

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

A microbe is defined as any microorganism (including both eukaryotic andprokaryotic organisms) such as fungi, yeasts, bacteria, actinomycetes,algae and protozoa, as well as other unicellular structures capable ofgrowth in culture.

A beauvericin-producing microbe is any microbe capable of producing themycotoxin beauvericin or analogs thereof. Such microbes are generallymembers of the fungal genus Fusarium, as well as recombinantly derivedorganisms which have been genetically altered to enable them to producebeauvericin or analogues thereof.

By degrading beauvericin, having the ability to degrade beauvericin, ismeant any modification or ability to make any modification to thebeauvericin molecule or a structurally related molecule that causes adecrease in or loss of its toxic activity. Such a change can comprisecleavage of any of the bonds, oxidation, reduction, the addition ordeletion of a chemical moiety, or any other change that affects theactivity of the molecule. Furthermore, chemically altered beauvericinmay be isolated from cultures of microbes that produce an enzyme of thisinvention, such as by growing the organisms on media containingradioactively-labeled beauvericin, tracing the label, and isolating thedegraded toxin for further study. The degraded beauvericin may becompared to the active compound for its phytotoxicity or mammaliantoxicity in known sensitive species, such as porcines.

By structurally related mycotoxin is meant any mycotoxin having achemical structure related to a beauvericin or analog of beauvericin, aswell as other mycotoxins having similar chemical structures that wouldbe expected to de detoxified by activity of the beauvericin degradativeenzymes.

Harvested grain is defined as any form of grain which has been somehowremoved from the environment in which it was grown. For example,harvested grain may comprise ear corn, or corn kernels, for example.Harvested grain may further comprise that in storage or that beingprocessed. Processed grain is grain that has been through some form ofprocessing and will be used in the production of food for humanconsumption or will be used as animal feed ("feed grain").

Within this application, plant refers to a photosynthetic organismincluding but not limited to an algae, moss, fern, gymnosperm, orangiosperm. Preferably, said plant is one from which feed grain(preferably for human or animal consumption) may be harvested("harvested grain"). Most preferably, said plant includes any variety ofcorn (maize), wheat, sorgum, rice and barley.

A mature plant is defined as a plant in which normal development of allvegetative and reproductive organs has occurred.

A plant cell includes any cell derived from a plant, including callus aswell as protoplasts, and embryonic and gametic cells.

The present invention comprises a methodology for the isolation of amicroorganism having the ability to degrade beauvericin or astructurally related mycotoxin, a microorganism having the ability todegrade beauvericin or a structurally related mycotoxin, and amethodology for degradation of beauvericin or a structurally relatedmycotoxin on a plant in the field as well as on harvested grain. Saidmicroorganism may include but is not limited to bacteria and fungi. Inorder to isolate said microorganism having the ability to degradebeauvericin or a structurally related mycotoxin, an assay was developedin which a microorganism is initially isolated from a source material.Said source material may comprise any plant or plant-associated materialincluding but not limited to any green tissue such as the stalk, leaf,ear, or kernel. Plant-associated material may include but is not limitedto soil in close approximation to the plant. Said microorganism is thencultured in media having beauvericin as the sole carbon source. Themedia is then observed for the disappearance of the beauvericin crystalsthat are initially present in said media prior to culturing saidmicroorganism in said media. The disappearance of said crystals isunderstood to indicate that said microroganism in said culture has theability to degrade beauvericin. The assay is termed a "crystaldisappearance" assay.

To test the ability of said microorganism isolated by theabove-described methodology to degrade or detoxify beauvericin or astructurally related mycotoxin on a plant, a mature plant is inoculatedwith a beauvericin-producing organism and then treated with anappropriate amount of bacteria having the ability to degrade or detoxifybeauvericin or its derivatives or analogs. The treatment may compriseapplication of a composition comprising an efficacious amount of anorganism having the ability to degrade beauvericin to said plant wherebythe beauvericin present is degraded. Preferably, said applicationconsists of topically applying said composition upon the tissues of saidplant, such that beauvericin upon said tissues is degraded.

Alternatively, said plant may be treated with said organism followingharvest (treatment of harvested grain). An important utility for thepresent invention is the detoxification of zearalenone present in grainfollowing harvest. A suitable feed material or "sample" is spiked with aknown amount of mycotoxin delivered in a suitable solvent, preferablyethanol, at an appropriate rate, preferably one ml solvent per gram,followed by sufficient mixing to distribute said mycotoxin throughoutsaid material. A control sample preferably receives solvent only. Thefinal concentration of said mycotoxin is preferably between 0.1 and 1.0mg per gram of feed material. The sample may then be air-dried to removeexcess solvent. The sample is next innoculated with 10⁵ -10⁷ colonyforming untis (cfu)/g of log-phase cells of a microorganism having theability to degrade said mycotoxin, at a sufficient rate, preferably oneml cells per gram, followed by sufficient mixing to distribute saidcells throughout said sample. A control sample may comprise cells thathave been killed by heating, preferably to approximately 80° C. Acontrol sample may further comprise cells of a microorganism that is notable to degrade said mycotoxin. Said samples are then placed into acontainer, said container is closed and incubated for a sufficientperiod of time at an appropriate temperature. Said period of time ispreferably within the range of one day to two weeks and said temperatureis preferably room temperature or approximately 28° C. Followingincubation, the contents of said container is extracted in a suitableorganic solvent (or organic aqueous mixture) for recovering saidmycotoxin. The resulting extract is then concentrated and subjected toqualitative and quantitative analysis for the presence of saidmycotoxin. The amount of said mycotoxin detected in said extract is thencompared to the amount of said mycotoxin detected in said controlsample, and the efficacy of removal of said mycotoxin expressed as apercent reduction in the level of said mycotoxin in said experimentalextract as compared to the level of said mycotoxin in said controlsample. In the instant invention, said mycotoxin is preferablybeauvericin. These methodologies allow for the degradation ofbeauvericin on or within said plant or harvested grain, thus providingimproved food grain quality and feed safety.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXAMPLE I

Isolation of Bacteria that Degrade Beauvericin

Various sources of plant material that were likely to naturally containbeauvericin were collected as source material for screening. Wheatkernels infested with Fusarium graminearum (F. graminearum; 140independent samples) were obtained from a Pioneer Hi-Bred International,Inc. ("Pioneer") wheat breeding station in Indiana. Silage samples wereobtained from the Microbial Genetics division of Pioneer and compostsamples from local residences (126 independent samples total).

The metabolism of beauvericin was measured using a crystal disappearanceassay. Microbes were washed from the source material by placing a smallamount in a seven milliliter Falcon tube and adding one to twomilliliters sterile distilled water (producing "wash fluid"). Maizekernels were split with a razor blade and one to two kernels were used.Tubes were capped and shaken for one to three hours at room temperature.Beauvericin (Sigma Cat. No. B7510) was prepared as a suspension inmineral salts medium, and was utilized as the sole carbon source. Thebeauvericin concentration utilized includes but is not limited to0.75-1.0 milligrams/milliliter in mineral salts medium. The mineralsalts medium was prepared by combination of reagents including but notlimited to 1.0 g/L ammonium sulfate, 1.0 g/L sodium chloride, 1.0 g/Lpotassium phosphate, dibasic, 0.2 g/L magnesium sulfate. Sterilizationof the solution was accomplished by filtration through a 0.2 micronfilter, although various methods for sterilization are available tothose skilled in the art. 100 microliters of beauvericin/mineral saltssuspension medium was added to each well of a microtiter plate (96 wellplate). One microliter of fresh wash fluid was added to each well.Control wells received one microliter of water. After two weeks, onemicroliter from each well was transferred to a new microtiter platecontaining 100 microliters of beauvericin/mineral salts medium. Thetransfer was then repeated four weeks later. After six weeks, wells werescored for partial disappearance of beauvericin crystals. Typically, thesmall crystals had been solubilized and metabolized, and only the verylargest beauvericin crystals remained. This effect was visualized usingan inverted microscope or by examining the plate visually from theunderside.

The instant invention comprises a biologically pure culture of amicroorganism responsible for beauvericin degradation. Saidmicroorganism was isolated using the following procedure. One microliterwas taken from positive wells and added to one milliliter of sterilewater. Several ten-fold dilutions were made in sterile water, and 100microliters from each dilution were plated and spread on YDP agarplates. YDP agar plates were prepared by combination of 10 grams yeastextract (Difco), 20 g Bacto peptone, 0.5 g dextrose, 15 g Bacto agar inwater followed by sterilization by autoclaving. From these mixtureculture spread plates, individual colonies were streaked for isolationon new YDP plates. An effort was made to choose at least one of everytype of bacteria represented on the spread plates. Each bacterium wasused to make a dilute suspension in sterile water, and one microliter ofthis suspension was used to inoculate microtiter wells containingbeauvericin in mineral salts as described above.

Initial characterization of bacteria was performed by Gram stainingsamples. more definitive identification was performed using acombination of techniques. Streak plates of individual bacterialcolonies were sent to Microbe Inotech Laboratories, Inc. (St. Louis,Mo.) for tentative identification. The analysis included camparison ofbacterial fatty acid methyl esters with Aerobe and Clinical Aerobedatabases, and Biolog™ substrate utilization comparison with a Grampositive database. Results of such tests have identified three speciesof Gram negative or Gram variable Nocardia globulera, Rhodococcusfascians, and Bacillus sphaericus and is demonstrated below in Table I.These cultures were deposited with the American Type Culture Collection(ATCC; 12301 Parklawn Drive, Rockville, Md. 20852 USA) on Oct. 15, 1996in accordance with the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.

                  TABLE 1                                                         ______________________________________                                        Microbial isolates having the ability to degrade beuavericin                    ATCC                 Tentative                                                                                  Number Name Identification Source         ______________________________________                                        55850  BEA(2)2904.G4                                                                             Nocardia globulera or                                                                       Wheat                                          kernals  Rhodococcus fascians  infested with                                     F. graminearum                                                             55849 BEA(1)2904.A12 Nocardia globulera or Wheat                              kernals  Rhodococcus fascians infested with                                      F. graminearum                                                             55848 BEA(1)2905.D1 Nocardia globulera or Wheat                               kernals  Bacillus sphaericus infested with                                       F. graminearum                                                             55847 BEA(1)2904.B11 Nocardia globulera or Wheat                              kernals  Rhodococcus infested with                                              erythropolis F. graminearum                                               ______________________________________                                    

EXAMPLE II Treatment of Beauvericin-contaminated Corn

A. Treatment of contaminated corn in the field

To test the ability of the bacteria isolated by the above-describedmethodology to degrade or detoxify beauvericin or its derivatives oranalogs on maize, mature plants are inoculated with abeauvericin-producing Fusarium sp. and then treated with an appropriateamount of bacteria having the ability to degrade or detoxify beauvericinor its derivatives or analogs. The treatment consists of topicallyapplying a composition comprising an efficacious amount of bacteria ontothe tissues of the maize plant such that beauvericin, including anyderivatives or analogs of beauvericin, is partially or completelydegraded or detoxified.

B. Treatment of contaminated corn after harvest.

A one to ten gram sample of cracked corn is combined or "spiked" with aknown amount of beauvericin in ethanol at a concentration of one grambeauvericin per ml of ethanol, followed by mixing to distribute thebeauvericin throughout the mixture. A control sample or samples aremixed with solvent alone. The sample is then air-dried to remove excesssolvent. The samples are then inoculated with 106 cfu/g of log-phasecells of a microorganism having the ability to degrade beauvericin,designated BEA(2)2904.G4 (deposited with the ATCC under accession numberATCC 55850) at a rate of one ml cells per gram, and mixed well todistribute said cells within said sample. Controls are mixed with eithercells of said microorganism [designated BEA(2)2904.G4, deposited withthe ATCC under ATCC accession number 55850] that have been heated to 80°C., such that said cells are non-viable or with cells of a microorganismthat does not have the ability to degrade beauvericin. Said mixture isplaced in a container, which is then closed and incubated for two weeksat room temperature. At the end of the incubation period, the containeris opened, and the entire contents extracted in a suitable organicsolvent to recover the beauvericin. The extract is concentrated andsubjected to qualitative and quantitative analysis for detection ofbeauvericin. The amount of beauvericin is determined and compared tocontrols. The efficacy of removal of beauvericin is determined bycomparison of the percent reduction (if any) of the amount ofbeauvericin in the sample comprising the micoorganism having the abilityto degrade beauvericin to the reduction of the amount of beauvericinpresent in said control sample. Microorganisms designated BEA(1)2904.A12(ATCC accession number 55849), BEA(1)2905.D1 (ATCC accession number55848), or BEA(1)2904.B11 (ATCC accession number 55847) are also able todegrade beauvericin, and may be utilized for the above-describedpurpose.

While a preferred form of the invention has been shown in the drawingsand described, since variations in the preferred form will be apparentto those skilled in the art, the invention should not be construed aslimited to the specific form shown and described, but instead is as setforth in the claims.

What is claimed is:
 1. An isolated bacterium which degrades beauvericinselected from the group consisting of a Nocardia species and aRhodococcus species.
 2. The bacterium of claim 1 wherein said bacteriumis Nocardia globerula.
 3. The bacterium of claim 1 wherein saidbacterium is Rhodoccus fascians.
 4. The bacterium of claim 1 whereinsaid bacterium is Rhodoccus erythropolis.